Improved methodologies for maximizing recombinant gene expression is an on-going effort in the art. Of particular interest is the development of methodologies that maximize recombinant expression of mammalian genes suitable for producing commercially useful quantities of biologically active proteins. While prokaryotic, typically bacterial, host cell systems have proven capable of generating large quantities of recombinant proteins, these hosts suffer from a number of disadvantages, including an inability to glycosylate proteins, inefficient cleavage of "pre" or "prepro" sequences from proteins (e.g., inefficient post translational modification), and a general inability to secrete proteins. Consequently the art has sought eukaryotic host systems, typically mammalian host cell systems, for mammalian protein production. One feature of such systems is that the protein produced has a structure most like that of the natural protein species, and, purification often is easier since the protein can be secreted into the culture medium in a biologically active form.
A number of problems still exist however, in mammalian culture systems. Specifically, high levels of expression typically are not easily obtained in mammalian systems. In addition, eukaryotic host cells typically have more stringent requirements for culturing and have slower growth rates. Thus, producing large quantities of a recombinant protein requires more than simply culturing a host cell transfected with an expression vector. This is particularly true when the gene of interest is a poorly expressed gene, i.e., is not produced in abundance or is only transiently expressed under natural, physiological conditions. The genes encoding these proteins typically have multiple levels of regulation, often at one or more levels of the expression system, e.g., at the level of transcription, translation, post translation modification, secretion and/or activation. Typically these genes, when stably integrated in unamplified, immortalized cells, produce less than about 10-100 ng protein/10.sup.6 cells per ml. Maximizing production of these proteins means identifying means for circumventing these levels of regulation.
Another class of poorly expressed DNA sequences are non-native, biosynthetic or otherwise artificial genes which contain one or more non-native DNA and/or RNA sequences or structures with which the host system is unfamiliar and which may limit and/or otherwise interfere with efficient protein production. Single chain binding proteins, also referred to in the art as "sFv" (single chain Fv) or "BABS" (biosynthetic antibody binding site) molecules are a particular example of such non-native DNA sequences. Typically, these constructs are expressed in E. coli, necessitating their purification from inclusion bodies and refolding in vitro. While prokaryotic cell secretion methods can obviate the need for refolding, there are numerous examples of particular sFv proteins that are not secreted from E. coli in active form, or do so at unacceptedly low levels. Moreover, it is not uncommon for secreted sFv molecules to form insoluble aggregates in the periplasm.
One approach to achieving enhanced protein production is use of transient cell expression systems wherein cells are transfected with high copy numbers of plasmids that are not expected to integrate in the host cell genome. The plasmids used in transient cell expression systems also can be modified to further enhance their copy numbers during replication post transfection. While the transfection event typically limits the life of these cells to only several generations, reasonable quantities of the desired protein may be produced while the cells remain alive. Because such transient cell systems are short-lived they are not cell systems of choice for commercial production systems. Transient cell systems often are used to screen candidate plasmid or other vector constructions as part of the development of an immortalized, constitutive cell line. But, because transient expression systems are short lived, the long-term productivity of a particular vector construction (or its effect, once integrated, on the viability of a cell after many generations) can not be determined with certainty. Accordingly, a number of plasmid constructions, while productive in transient cell systems, have been determined not to be useful in established cell lines, an event that generally cannot be determined until an established cell line is created.
Two alternative ways primarily focused on by the art for enhancing recombinant gene expression in eukaryotic host systems are enhancing the gene copy number, typically by gene amplification, and enhancing the efficiency of expression of each gene copy. The most common method for enhancing gene copy number is by selecting for gene amplification wherein the host cell is transformed with two genes, linked or unlinked, one of which encodes the desired protein and the other of which encodes an amplifiable selectable marker, such as dihydrofolate reductase (DHFR.) Transformed cells then are cultured in the presence of increasing concentrations of a toxic agent (e.g., methotrexate, when the amplifiable marker is DHFR) whose effects can be nullified by expression of the selectable marker gene. In response to high concentrations of the toxic agent cells survive because they have amplified the copy number of the selectable marker gene and, fortuitously, the desired protein gene. Using this methodology copy numbers in the hundreds and thousands/cell have been achieved.
While gene amplification has proven to be useful, the methodology suffers from several disadvantages pertinent to commercial production. For example, the production of a highly productive cell line by gene amplification alone, e.g., having thousands of copies of the gene of interest, is a time-consuming process often requiring between 6-10 months to complete. Moreover, at very high copy number, verification of the nucleotide sequence integrity for each gene copy in a cell is difficult or not possible. Accordingly, point mutations and other sequence modifications that can alter the biological activity of the protein product may not be detected, and further may pose problems with compliance of government (e.g., FDA) regulations. Moreover, maintenance of such a high copy number requires maintaining the selective pressure by maintaining high levels of the toxic agent in the culture medium. This is both expensive and presents additional regulatory issues when purifying the protein of interest from the culture medium. Finally, and perhaps most importantly, when a gene has multiple levels of expression regulation, merely increasing the copy number of the DNA may not be sufficient to enhance protein production significantly.
One method for enhancing recombinant DNA expression is by means of one or more genes encoding expression effector molecules. Among the effector molecules known in the art are transacting transcription activators which can stimulate transcription of heterologous genes. Examples include the simian virus (SV40) T antigen and adenovirus E1A and E1B proteins which can act on certain viral promoters of heterologous genes, including the cytomegalovirus (CMV) major intermediate early (MIE) promoter. Other molecules reported to have this transactivating activity include the immediate early (IE) proteins of herpes virus, C-myc and genes of the human and simian acquired immunodeficiency virus.
Other viral genes which can effect mammalian protein production are viral translational control effectors, including genes encoding RNA sequences operative to promote translation. Examples include RNA sequences encoded by the adenovirus, such as the VA genes (VA1 and VA2). Such sequences are believed to assist protein production by assisting with translation initiation, probably by association with one or more translation initiation factors. Other examples include RNA sequences that stabilize the transcript.
Cockett et al., ((1990) Nucleic Acids Research 19:319-325 and EP application 378,382) describe the use of the adenovirus E1A genes as an alternative to gene amplification for recombinant protein expression in Chinese hamster ovary (CHO) cells, where the gene of interest is under the CMV promoter control. The level of protein produced is asserted to approach levels achievable by gene amplification, thereby obviating the need for gene amplification. Moreover, the authors see no substantial increase in protein productivity when the E1A gene is introduced to an amplified cell line expressing the gene of interest.
U.S. Pat. No. 5,024,939 describes an unamplified transient cell expression system producing "useful" quantities of a desired gene product in 1 to 14 days without having to establish a continuous production cell system. The authors transfect E1A-expressing cells ("293" cells) with a large number of plasmids carrying the gene of interest under CMV promoter control, and demonstrate increased protein production in these cells for the short lives of the cells. Co-transfection of the 293 cells with the adenovirus VA1 gene appears to double the amount of protein produced in these cells.
It is an object of the instant invention to provide a method for enhancing protein production of poorly-expressed non-native DNA sequences by recombinant DNA technology. It is another object of the invention to provide immortalized cell lines suitable for commercial exploitation wherein the cells are stably transfected with the non-native gene of interest and are competent to constitutively express the gene of interest, and to provide methods for producing these cell lines. Still another object of the invention is to provide cell lines, and methods for creating them, exhibiting high non-native recombinant protein productivity while maintaining a low copy number per cell of the recombinant non-native DNA sequences encoding the protein. Yet another object is to provide cell lines that can be adapted to grow in low serum or serum-free medium.
Importantly, it is another object of the instant invention to provide means for producing commercially-feasible quantities of mono- and bifunctional single-chain antibody binding site constructs (sFv) from cultures of immortalized, stably transfected CHO cell lines.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.